MX2007012336A - Multivalent pneumococcal polysaccharide-protein conjugate composition. - Google Patents

Abstract

An immunogenic composition having 13 distinct polysaccharide-protein conjugates and optionally, an aluminum-based adjuvant, is described. Each conjugate contains a capsular polysaccharide prepared from a different serotype of Streptococcus pneumoniae (1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F) conjugated to a carrier protein. The immunogenic composition, formulated as a vaccine, increases coverage against pneumococcal disease in infants and young children globally, and provides coverage for serotypes 6A and 19A that is not dependent on the limitations of serogroup cross-protection.

Description

COMPOSITION OF PROTEIN CONJUGATES-MULTISTABLE POLYACCHARIDES FOR PNEUMOCOCES FIELD OF THE INVENTION The present invention relates generally to the field of medicine, and specifically to microbiology, imlogy, vaccines and the prevention of infection by a bacterial pathogen by imzation.

BACKGROUND OF THE INVENTION Streptococcus pneumoniae is an important cause of meningitis, pneumonia, and severe invasive disease in newborns and young children throughout the world. Multivalent pneumococcal polysaccharide vaccines have been licensed for several years and have proven valuable in the prevention of pneumococcal disease in older adults and high-risk patients. However, newborns and young children respond poorly to most pneumococcal polysaccharides. The heptavalent pneumococcal conjugate vaccine (7vPnC, Prevnar®) was the first of its kind to be highly imgenic and effective against invasive disease and otitis media in newborns and young children. This vaccine is now approved in many countries around the world. Prevnar contains the polysaccharides in casual of serotypes 4, 6B, 9V, 14, 18C, Ref .: 186704 19F and 23F, each conjugated to the carrier protein designated CRM197. Prevnar covers approximately 80-90%, 60-80%, and 40-80% of invasive pneumococcal disease (IPD) in the United States, Europe, and other regions of the world, respectively [1,2]. Research data collected in the years following the introduction of Prevnar have clearly demonstrated a reduction of invasive pneumococcal disease in newborns from the United States. as expected (FIG.1) [3,]. The investigation of the IPD carried out in newborns of the U.S. prior to the introduction of Prevnar showed that a significant portion of the disease due to serogroups 6 and 19 was due to serotypes 6A (approximately one third) and 19A (approximately one quarter) [5,6]. The investigation of invasive pneumococcal disease carried out in the United States. after the licensing of Prevnar suggests that a large burden of the disease is still attributed to serotypes 6A and 19A (FIG 1) [3]. Additionally, these two serotypes account for the majority of cases of invasive disease that serotypes 1, 3, 5, and 7F combined (8.2 vs. 3.3 cases / 100, 000 children 2 years and younger). In addition, serotypes 6A and 19A are associated with high rates of antibiotic resistance (Figure 2) [7,8,9]. Although it is possible that cross protection to the serogroup will result in a decline of the disease by the serotype 6A and 19A as more children become imzed, there is evidence to suggest that there will be a limit to the decline, and a significant burden of disease will remain due to these serotypes (see below). Given the relative burden and importance of invasive pneumococcal disease due to serotypes 1, 3, 5, 6A, 7F, and 19A, the addition of these serotypes to the Prevnar formulation will increase the coverage of invasive disease up to > 90% in the US and Europe, and as high as 70% -80% in Asia and Latin America. This vaccine will significantly expand coverage beyond that of Prevnar, and provide coverage for 6A and 19A that does not depend on the limitations of cross-protection of serogroups.

BRIEF DESCRIPTION OF THE INVENTION In this manner, the present invention generally provides a multivalent imgenic composition comprising 13 different polysaccharide-protein conjugates, wherein each of the conjugates contains a capsular polysaccharide from a different serotype of Streptococcus pneumoniae conjugated to a carrier protein, together with a physiologically acceptable carrier. Optionally, an adjuvant, such as an aluminum-based adjuvant, is included in the formulation. More specifically, the present invention provides a composition of a conjugate of 13-valent pneumococcus (13vPnC) comprising the seven serotypes in the 7vPnC vaccine (4, 6B, 9V, 14, 18C, 19F and 23F) plus six additional serotypes (1, 3, 5, 6A, 7F and 19A). The present invention also provides a multivalent immunogenic composition, wherein the capsular polysaccharides are of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae and the protein carrier is CRM197. The present invention further provides a multivalent immunogenic composition, wherein the capsular polysaccharides are of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9v, 14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae, the protein carrier is CRM197, and the adjuvant is an aluminum-based adjuvant, such as aluminum phosphate, aluminum sulfate and aluminum hydroxide. In a particular embodiment of the invention, the adjuvant is aluminum phosphate. The present invention also provides a multivalent immunogenic composition, comprising polysaccharide-protein conjugates together with a physiologically acceptable carrier, wherein each of the conjugates comprises a capsular polysaccharide of a different serotype of Streptococcus pneumoniae conjugated to a carrier protein, and Capsular polysaccharides are prepared from serotype 3 and at least one additional serotype. In one embodiment of this immunogenic composition multivalent, the additional serotype is selected from the group consisting of serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F. In another embodiment, the carrier protein is CRM? 97. In yet another embodiment, the composition comprises an adjuvant, such as an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide. In a particular embodiment, the adjuvant is aluminum phosphate. The present invention also provides a multivalent immunogenic composition, comprising polysaccharide-protein conjugates together with a physiologically acceptable carrier, wherein each of the conjugates comprises a capsular polysaccharide of a different serotype of Streptococcus pneumoniae conjugated to a carrier protein, and Capsular polysaccharides are prepared from serotypes 4, 6B, 9V, 14, 18C, 19F, 23F and at least one additional serotype. In one embodiment of this multivalent immunogenic composition, the additional serotype is selected from the group consisting of serotypes 1, 3, 5, 6A, 7F, and 19A. In another embodiment, the carrier protein is CRM197. In yet another embodiment, the composition comprises an adjuvant, such as an aluminum-based adjuvant selected from aluminum phosphate, aluminum sulfate and aluminum hydroxide. In a particular embodiment, the adjuvant is aluminum phosphate.

The present invention also provides a method for inducing an immune response to a capsule polysaccharide conjugate of Streptococcus pneumoniae, which comprises administering to a human, an immunologically effective amount of any of the immunogenic compositions described above. The present invention further provides that any of the immunogenic compositions administered is a single dose of 0.5 mL formulated to contain: 2 μg of each saccharide, except for 6B to 4 μg; approximately 29 μg of carrier protein CRM197; 0.125 mg of elemental aluminum adjuvant (0.5 mg of aluminum phosphate); and sodium chloride and sodium succinate buffer solution as excipients.

BRIEF DESCRIPTION OF THE FIGURES FIG. 1 details the changes in the IPD relationships for the serotype in E children. OR . of < 2 years old from the baseline (1998/1999) to 2001. FIG. 2 details the distribution of pneumococcal isolates with penicillin resistance (PCN) in children of < 5 years old (1998). FIG. 3 details the reverse distribution curves (RCDC) of the results of the dose after the third dose of OPA from the Prevnar test D118-P16.

DETAILED DESCRIPTION OF THE INVENTION Inclusion of Servotypes of Prevn &r 4, SB, 9V, 14, 18C, 19F, 23F Data from the IPD research between 1995-1998 estimated that the seven serotypes in Prevnar were responsible for about 82% of IPD in children of < 2 years old [5]. In northern California, the site of the efficacy trial, Prevnar serotypes accounted for 90% of all cases of IPD in newborns and young children [10]. Since the introduction of the Prevnar vaccine in 2000, there has been a significant decline in global IPD ratios due to the decrease in disease due to vaccine serotypes [3,4]. Therefore, there is no justification at this time to remove some of the serotypes of Prevnar from the next generation of pneumococcal conjugate vaccines but rather to add serotypes to obtain greater coverage.

Inclusion of Serotypes 1, 3, 5 and 7F In the United States, the IPD ratio caused by serotype 1 in children under 5 years of age is < 2%, around the same as for each of the types 3 and 7F [1, 6]. Serotypes 1 and 5 represent the highest IPD ratios in US populations. with high risk of disease due to invasive pneumococcus. Specifically, the serotype 1 causes 3.5% of IPD in Alaska Native children of < 2 years of age, and 18% in children 2-4 years of age [11].

Both serotype 1 and serotype 5 cause significant disease in other parts of the world and in indigenous populations in developed countries [12,13,14].

Serotype 1 can also be associated with more severe disease compared to other pneumococcal serotypes [fifteen] . This observation is based on the difference in case identification relationships between the US. and Europe, and the associated difference in medical practice. In general, the incidence of IPD is lower in Europe than in the US. However, the percentage of IPD caused by serotype 1 in Europe is disproportionately higher than in the US. (6-7%, vs 1-2%, respectively). In Europe, blood cultures are obtained mainly from hospitalized children. In the U.S., it is routine medical practice to obtain blood cultures in a non-patient setting from children presenting with > 39 ° C and high white blood cell counts. Given the difference in medical practice, it is postulated that the lower percentage of disease caused by serotype 1 in the US. it can be diluted by higher ratios of other serotypes that cause a more moderate disease, while the higher percentage in Europe reflects a more serious disease. In addition, studies of seroepidemiology of children with Complicated pneumonia show that serotype 1 is disproportionately represented [16,17,18]. This suggests that the inclusion of serotype 1 can reduce the amount of severe pneumococcal disease, as well as contribute to the total reduction in invasive pneumococcal disease. The addition of serotypes 3 and 7F will increase coverage against IPD in most areas of the world by approximately 3% -7%, and in Asia by around 9%. Thus, an 11-valent vaccine will cover 50% in Asia and around 80% of the IPD in all other regions [1,2]. These serotypes are also important with respect to otitis media coverage [19]. In a multinational study of pneumococcal serotypes that cause otitis media, Hausdorff et al. Found that serotype 3 was the 8th. isolated from the most common middle ear fluid in general [20]. Serotype 3 represented up to 8.7% of the pneumococcal serotypes associated with otitis media. Thus, the importance of types 3 and 7F in otitis media, as well as in IPD, guarantees their inclusion in the pneumococcal conjugate vaccine. However, efforts to produce a vaccine of a multivalent pneumococcal conjugate that shows significant immunogenicity with respect to serotype 3 polysaccharides has not been successful. For example, in a study of the immunogenicity and safety of the vaccine of the 11-valent conjugate of protein D of pneumococci (11-Pn-PD), no observed no primer effect for serotype 3 in newborns who had received three doses of the vaccine followed by a booster dose of either the same vaccine or a polysaccharide from the pneumococcal vaccine (Nurkka et al. (2004) Ped. Dis. J., 23: 1008-1014). In another study, the results of the opsonophagocytic (OPA) trial of children who had received doses of 11-Pn-PD failed to show antibody responses for serotype 3 at levels comparable to other serotypes tested (Gatchalian et al., 17th Meeting Annual Eur. Soc. Paed, Inf. Dis. (ESPID), Poster No. 4, PIA Poster Session 1, Istanbul Turkey, Mar. 27, 2001). In yet another study, which evaluated the efficacy of 11-Pn-PD in the prevention of acute otitis media, the vaccine did not provide protection against episodes caused by serotype 3 (Prymula et al., Www.thelancet.com. Vol. 367: 740-748 (March 4, 2006)). Thus, a vaccine of a pneumococcal conjugate comprising capsular polysaccharides of serotype 3 and which can produce an immunogenic response to the polysaccharides of serotype 3 provides a significant improvement over the existing state of the art.

Inclusion of Serotypes 6A and 19A a. Epidemiology of Serotypes 6A and 19A Research data in the literature suggest that serotypes 6A and 19A represent more invasive pneumococcal disease in children of E.U. of < 2 years of age serotypes 1, 3, 5, and 7F combined (FIG 1) [1,5]. In addition, these serotypes are commonly associated with resistance to antibiotics (FIG 2) and play an important role in otitis media [6,19,20]. The ability of the current Prevnar vaccine to protect against the disease due to 6A and 19A is unclear. The rationale for the inclusion of components 6A and 19A in a 13vPnC vaccine are discussed below. b. Responses to 6A and 19A Induced by Polysaccharides 6B and 19F Licensed vaccines of unconjugated pneumococcal polysaccharide (for use in persons at least two years of age) have contained capsular polysaccharide 6A or 6B but not both [21]. The immunogenicity data generated at the time of the formulation of the 23-valent pneumococcal polysaccharide vaccine demonstrated that a monovalent 6B vaccine induced antibodies to both capsules 6A and 6B. Data from several trials evaluating IgG responses and the opsonophagocytic assay (OPA) in a variety of populations with free polysaccharide and with pneumococcal conjugate vaccine suggest that IgG responses to 6A are induced by 6B antigens, but answers with generally lower, and OPA activity with 6A organisms is different than with 6B organisms [22,23,24,25], In addition, subjects that respond with a high 6B antibody may have little or no activity against 6A. In contrast to the chemical composition of capsular polysaccharides 6A and 6B where there is a high degree of similarity, capsules 19A and 19F are quite different due to the presence of two additional side chains in polysaccharide 19A. Not surprisingly, the immune responses measured in human volunteers immunized with the 19F polysaccharide vaccine showed that responses to 19F were induced in 80% of the subjects, but only 20% of the subjects had a response to 19A [26]. Low levels of cross-reactive IgG and OPA responses to serotype 19A after immunization with 19F polysaccharide have also been documented in conjugate vaccine trials [24,26]. The internal data of the cross-reactive responses of OPA to 6A and 19A were generated from the 7vPnC bridging test (D118-P16) carried out in newborns of E.U. (FIG 3) These studies consistent with the findings of others, and demonstrate the induction of functional cross-reactive antibody to polysaccharide 6A after immunization with polysaccharide 6B, albeit at a lower level, and very little functional antibody to 19A after the immunization with 19F.

Impact of Immunization of 6B and 19F on 6A and 19A in Animal Models Animal models have been used to evaluate the potential for cross protection with polysaccharide immunization. In an otitis media model developed by Giebink et al., Chinchillas were immunized with a tetravalent polysaccharide outer membrane protein conjugate vaccine (OMP) (containing saccharides 6B, 14, 19F, 23F) or placebo [27]. In this trial there seemed to be some cross-protection for 6A; however, this did not reach statistical significance and the level of protection was lower than with 6B against otitis media. In this same model, there was 100% protection against otitis media for 19F, but only 17% protection against otitis media for 19A. Saeland et al. used sera from newborns immunized with an 8-valent pneumococcal tetanus conjugate vaccine (containing 6B and 19F) to passively immunize mice prior to an intranasal immunogenic test with 6A organisms, in a model of infection in the lung [28]. ] Of the 59 serum samples, 53% protected the mice against bacteremia with 6B and 37% protected against 6A. The mice passively immunized with sera of newborns immunized with four doses of a vaccine of 11-valent pneumococcal conjugate (containing 19F conjugated to tetanus toxoid) was given an intranasal immunogenic test with 19A organisms in the same model [29]. Of the 100 mice passively immunized and then applied with immunogenic test, 60 mice had no 19A organisms detected in the lung tissue, while organisms were identified in all mice given saline placebo. However, passive immunization did not protect against the immunogenic test with 19F organisms in this model; therefore, the relevance of the model for serogroup 19 is questionable. In general, these models provide evidence of some biological impact of 6B immunization on 6a organisms, although the effect of the heterologous serotype was not as great as that observed with the homologous serotype. The impact of 19F immunization on organisms 19A was not well understood from these models.

Impact of Immunization of Polysaccharide Conjugate 63 and 19F on Disease by 6A and 19A in Efficacy / Efficiency Trials The number of cases of disease due to serotypes 6B, 6A, 19F and 19A in the efficacy trials of 7vPnC and 9vPnC (7vPnC plus serotypes 1 and 5) is indicated in Table 1 [30,10,31]. The numbers of cases of invasive disease are too small to allow drawing some conclusions from serotypes 6A and 19A. However, the Finnish trial of otitis media generated a large number of pneumococcal isolates [32]. In the analysis by protocol 7vPnC was 84% (95% Cl 62%, 93%) effective against otitis media due to serotype 6B and 57% (95% Cl 24%, 76%) effective against otitis media due to serotype 6A (Table 1) . In contrast, the specific efficacy of the serotype with 7vPnC was not demonstrated for otitis media due to either 19F or 19A.

Table 1. Cases of Pneumococcal Disease Due to Serotypes 6B, 6A, 19F, and 19A in 7vPnC and 9vPnC Vaccine Efficacy Tests * Statistically significant efficacy demonstrated From references 30, 10 and 33, and personal communications Contr = ITT control = intention to treat PP analysis = analysis per protocol IPD marketing post-marketing research data are also available from a case control trial carried out by the Centers for Disease Control to evaluate the effectiveness of Prevnar [33]. The cases of invasive pneumococcal disease that occur in children from 3 to 23 months of age were identified in the research laboratories and coincided with three cases of control by age and zip code. After obtaining consent, medical and immunization history was obtained (subjects were considered immunized if they had received at least one dose of Prevnar) from parents and medical providers for cases and controls. Preliminary results were presented at the 2003 ICAAC meeting and a summary of the findings for the 6B disease, 19F, 19A and 6A are presented in Table 2. These data indicate that Prevnar can avoid disease due to 6A, although at a level that may be somewhat lower than the disease of serotype 6B. These data also indicate that cross protection for invasive disease due to 19A is limited.

Table 2. Preliminary Results of a Case Control Trial Carried out by the CDC (presented at the ICAAC 2003) * Effectiveness of the Vaccine when comparing vaccinates (> 1 dose) vs. unvaccinated, and adjusted for underlying conditions Reference 40 and personal / confidential communication A published analysis [3] of the use of Prevnar also indicated that serotypes 6B and 19F conferred a moderate reduction in IPD caused by serotypes 6A and 19A among children under of two years of age (Table 1 in [3]). Disease relationships among non-immunized adults caused by serotypes 6A, 9A, 9L, 9N, 18A, 18B, 18F, 19A, 19B, 19C, 23A and 23B ("all serotypes related to vaccines") were reduced by some way (Table 2 in [3]). These data establish that the immunity of the herd from the use of Prevnar in children under two years of age was modest for serotypes 6A and 19A, and provides a basis for for the inclusion of serotypes 6A and 19A in the 13vPnC vaccine of this invention.

Conclusion for the addition of 6A and 19A The post-marketing research data and the results of the case control study outlined in FIG. 1 and Table 2 with the 7vPnC vaccine suggest that, consistent with the other information in the immune responses and performance in the animal models described above, there may be some cross-protection against the 6A disease, but to a lesser extent than for the disease by 6B. Additionally, it seems that the protection against 19A is limited. Therefore, a 13vPnC vaccine containing serotypes 6A and 19A provides coverage that is not dependent on the limitations of serogroup cross protection by serotypes 6B and 19F. Accordingly, the present invention provides a multivalent immunological composition comprising 13 different protein-polysaccharide conjugates, wherein each of the conjugates contains a capsular polysaccharide conjugate different from a carrier protein, and wherein the capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae, together with a physiologically acceptable carrier. One such carrier protein is diphtheria toxoid designated CRM197. The immunogenic composition may further comprise an adjuvant, such as an aluminum-based adjuvant, such as aluminum phosphate, aluminum sulfate and aluminum hydroxide. The capsular polysaccharides are prepared by standard techniques known to those skilled in the art. In the present invention, capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F of Streptococcus pneumoniae. These pneumococcal conjugates are prepared by separate processes and formulated in a single dose formulation. For example, in one embodiment, each pneumococcal polysaccharide serotype is grown in a soy-based medium. The individual polysaccharides are then purified by centrifugation, precipitation, ultrafiltration, and column chromatography. The purified polysaccharides are chemically activated to make the saccharides capable of reacting with the carrier protein. Once activated, each capsular polysaccharide is separately conjugated to a carrier protein to form a glycoconjugate. In one embodiment, each capsular polysaccharide is conjugated to the same carrier protein. In this modality, the conjugation is effected by reductive amination. The chemical activation of polysaccharides and conjugation after the carrier protein are reached by conventional means. See, for example, Pat. From U.S.A. Nos. 4,673,574 and 4,902,506 [34,35]. The carrier proteins are preferably proteins that are non-toxic and non-reactive and obtainable in sufficient quantity and purity. Carrier proteins should be favorable to standard conjugation procedures. In a particular embodiment of the present invention, CR 197 is used as the carrier protein. CRM197 (Wyeth, Sanford, NC) is a non-toxic variant (ie, toxoid) of diphtheria toxin isolated from cultures of C7 strain Corynebacterium diphtheria (ßl97) grown in casamino acids and medium based on yeast extract. CRM197 is purified through ultrafiltration, ammonium sulfate precipitation, and ion exchange chromatography. Alternatively, CR 197 is prepared recombinantly in accordance with the Patent of E.U.A. No. 5,614,382, which is incorporated herein by reference. Other diphtheria toxoids are also suitable for use as carrier proteins. Other suitable carrier proteins include inactivated bacterial toxins such as tetanus toxoid, pertussis toxoid, cholera toxoid (for example, as described in International Patent Application WO2004 / 083251 [38]), E. coli LT, E. coli ST, and Pseudomonas exotoxin A aerugmosa Bacterial outer membrane proteins such as outer membrane complex c (OMPC), porins, transferrin-binding proteins, pneumolis, pneumococcal surface protein A (PspA), pneumococcal adhesin protein (PsaA), C5a peptidase of Group streptococci A or Group B, or protein D of Haemophilus mfluenzae, can also be used. Other proteins, such as ovalbumma, hemocyanin of a variety of limpet (KLH), bovine serum albumin (BSA) or purified protein derived from tuberculm (PPD) can also be used as carrier proteins. After conjugation of the capsular polysaccharide to the carrier protein, the polysaccharide protein conjugates are purified (enriched with respect to the amount of protein-polysaccharide conjugate) by a variety of techniques. These techniques include concentration / diafiltration, precipitation / elution, column chromatography, and deep filtration operations. See examples below. After the individual glycoconjugates are purified, they are compounded to formulate the immunogenic composition of the present invention, which can be used as a vaccine. The formulation of the immunogenic composition of the present invention can be made using methods recognized in the art. For example, the 13 individual pneumococcal conjugates can be formulated with a vehicle physiologically acceptable to prepare the composition. Examples of such carriers include, but are not limited to, water, buffered saline, polyols (eg, glycerol, propylene glycol, liquid polyethylene glycol) and dextrose solutions. In certain embodiments, the immunogenic composition will comprise one or more adjuvants. As defined herein, an "adjuvant" is a substance that serves to enhance the immunogenicity of an immunogenic composition of this invention. Thus, adjuvants are frequently given to elevate the immune response and are well known to those skilled in the art. Suitable adjuvants for increasing the effectiveness of the composition include, but are not limited to: (1) aluminum (alum) salts, such as aluminum hydroxide, aluminum phosphate, aluminum sulfate, etc .; (2) oil-in-water emulsion formulations (with or without other specific immunostimulatory agents such as muramyl peptides (defined below) or bacterial cell wall components), such as, for example, (a) MF59 (PCT Publ. No. WO 90/14837), which contains % Squalene, 0.5% Tween 80, and 0.5% Span 85 (optionally they contain different amounts of MTP-PE (see below, although not required)) is formulated into submicron particles using such a microfluidizer as Microfluidizer Model HOY (Microfluidics, Newton, MA), (b) SAF, containing 10% Squalene, 0.4% Tween 80, 5% Polymer L121 blocked with pluronic, and thr-MDP (see below) is either microfluidized in a submicron emulsion or placed in vortex to generate an emulsion of larger particle size, and (c) Ribi ™ adjuvant system (RAS), (Corixa, Hamilton, MT) containing 2% Squalene, 0.2% Tween 80, and one or more bacterial cell wall components of the group consisting of 3-O-dealiated monophosphorylid A (MPL ™) described in U.S. Patent No. 4,912,094 (Corixa), trehalose dimycolate (TDM), and cell wall skeleton (CWS), preferably MPL + CWS (Detox ™); (3) saponin adjuvants, such as Quil A or STIMULON ™ QS-21 (Antigenics, Framingham, MA) (U.S. Patent No. ,057,540) can be used or particles generated from this such as ISCOMs (immunostimulatory complexes); (4) bacterial lipopolysaccharides, synthetic lipid A analogs such as aminoalkyl glucosamine phosphate compounds (AGP), or derivatives or analogs thereof, which are available from Corixa, and which are described in the U.S. Patent No. 6,113,918; one of such AGP is 2 - [(R) -3-Tetradecanoyloxytetradecanoylamino] ethyl 2-Deoxy-4-0-phosphono- 3-0- [(R) -3-tetradecanoyloxytetradecanoyl] -2- [(R) -3-tetradecanoyloxytetradecanoylamino] -bD-glucopyranoside, which is also known as 529 (formerly known as RC529), which is formulated as an aqueous form or as a stable emulsion, synthetic polynucleotides such as oligonucleotides containing CpG portions (US Patent No. 6,207,646); (5) cytokines, such as interleukins (e.g., IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-12, IL-15, IL-18, etc.) ), interferons (eg, gamma interferon), granulocyte macrophage colony stimulating factor (GM-CSF), macrophage colony stimulating factor (M-CSF), factor necrosis factor (TNF), co-molecules stimulators B7-1 and B7-2, etc.; (6) detoxified mutants of a bacterial ADP ribosylated toxin such as a cholera toxin (CT) either in a wild type or mutant form, for example, where the amino acid-29 glutamic acid is replaced by another amino acid, preferably a histidine, in accordance with published international patent application number WO 00/18434 (see also WO 02/098368 and WO 02/098369), a pertussis toxin (PT), or a heat unstable toxin E. col i (LT), particularly LT-K63, LT-R72, CT-S109, PT-K9 / G129 (see, for example, WO 93/13302 and WO 92/19265); and (7) other substances that act as immunostimulatory agents to increase the effectiveness of the composition Muramyl peptides include, but are not limited to, N-acetyl-muramyl-L-threonyl-D-isoglutamine (thr-MDP), N-acetyl normuramyl-L-alanine-2- (1 '-2' dipalmitoyl) -sn-glycero-3-hydroxyphosphoryloxy) -ethylamine (MTP-PE), etc. The vaccine formulations of the present invention can be used to protect or treat a human susceptible to pneumococcal infection, by administering the vaccine via a systemic or mucosal route. These administrations may include injection via intramuscular, intraperitoneal, intradermal or subcutaneous routes; or by means of mucosal administration for oral feeding, respiratory or genitourinary tracts. In one embodiment, intranasal administration is used for the treatment of pneumonia or otitis media (as a pneumococcal nasopharyngeal transporter it can be prevented more effectively, thus relieving the infection at its early stage). The amount of conjugate in each vaccine dose is selected as an amount that induces a minor immunoprotective response, adverse effects. Such amount may vary depending on the pneumococcal serotype. Generally, each dose will comprise 0.1 to 100 μg of polysaccharide, particularly 0.1 to 10 μg, and more particularly 1 to 5 μg. The optimal amounts of the components for a Particular vaccine can be verified by standard studies that involve the observation of appropriate immune responses in subjects. After an initial vaccination, subjects may receive one or several differently spaced booster injection immunizations. In a particular embodiment of the present invention, the 13vPnC vaccine is a sterile liquid formulation of pneumococcal capsular polysaccharides of serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F individually conjugated to CRM? 97. Each 0.5 mL dose is formulated to contain: 2 μg of each saccharide, except for 6B to 4 μg; approximately 29 μg of carrier protein CRM197; 0.125 mg of elemental aluminum adjuvant (0.5 mg aluminum phosphate); and sodium chloride and sodium succinate buffer solution as excipients. The liquid is filled in single-dose syringes without a preservative. After shaking, the vaccine is a white, homogenous suspension, ready for intramuscular administration. The choice of dose level for the 13vPnC vaccine is similar to the 7vPnC vaccine marketed (Prevnar). The 2 μg saccharide dose level was selected for all serotypes, except for 6B, which is 4 μg per dose. The 7vPnC vaccine has shown safety, immunogenicity, and desirable efficacy against IPD at the 2μg saccharide dose level for serotypes 4, 9V, 14, 18C, 19F and 23F, and at the 4μg dose for 6B. The immunization program can follow the design for the 7vPnC vaccine. For example, the routine program for infants and children against invasive disease caused by S. pneumoniae due to the serotypes included in the 13vPnC vaccine is 2, 4, 6 and 12-15 months of age. The compositions of this invention are also suitable for use with older children, adolescents and adults. The compositions of this invention may also include one or more additional antigens for use against otitis media caused by infection with another bacterium. Such bacteria include Haemophilus infl uenza unclassifiable, Moraxella ca tarrha lis (formerly known as Branhamella ca tarrhal is) and Alloiococcus oti tidis. Examples of non-classifiable Haemophilus infl uenzae antigens for inclusion include protein P4, also known as "e" protein (U.S. Patent No. 5,601,831, International Patent Application WO 03/078453), protein P6, also known as the PAL protein or the PBOMP-1 protein (US Patent No. 5,110,908, International Patent Application WO 0100790), the P5 protein (US Patent No. 37,741), the adhesion of Haemofilus and penetration protein (patent of USA Nos. 6,245,337 and 6,676,948), the LKP limb ahesin protein (US Patent No. 5,643,725) and the NucA protein.

(U.S. Patent No. 6,221,365). Examples of Moraxel antigens appropriate for inclusion include the UspA2 protein (U.S. Patent Nos. 5,552,146, 6,310,190), the CD protein (U.S. Patent No. 5,725,862), the E protein (U.S. Patent No. 5,948,412) and the 74 kilodalton outer membrane protein (US Patent No. 6,899,885). Examples of antigens Alloiococcus oti tidis suitable for inclusion include those identified in International Patent Application WO 03/048304. The compositions of this invention may also include one or more Streptococcus pneumoniae proteins. Examples of Streptococcus pneumomae proteins suitable for inclusion include those identified in International Patent Application WO02 / 083855, as well as described in International Patent Application WO 02/053761.

The compositions of this invention may further include one or more Neissepa meningi tidis type B proteins. Examples of Nei sseria memngi tidis type B proteins appropriate for inclusion include those identified in International Patent Applications WO 03/063766, WO 2004 / 094596, WO 01/85772, WO 02/16612 and WO 01/87939. The foregoing description generally describes the present invention. A more complete understanding can be obtained by reference to the following specific examples.

These examples are described only for the purpose of the illustration and are not intended to limit the scope of the invention.

EXAMPLES Example 1 Preparation of Serotype 1 of Capsular Polysaccharide of S. Pne? Moniae Preparation of Master and Working Cell Bank Serotype 1 of S. pneumoniae was obtained from the American Type Culture Collection, ATCC, strain 6301. Several generations of reserve seeds were created in order to expand the strain and remove the components of animal origin (generations Fl, F2, and F3). Two additional generations of reserve seeds were produced. The first additional generation was made from an F3 vial, and the subsequent generation was made from a vial of the first additional generation. The vials of the seed were stored by freezing (< -70 ° C) with synthetic glycerol as a cryopreservator. In addition to frozen vials, lyophilized vials were prepared for the F4 generation. For the preparation of the cell bank, all the cultures were grown in a soy-based medium. Before freezing, the cells were concentrated by centrifugation, the spent medium was removed, and the cell pellets were re- suspended in fresh medium containing a cryopreservator, such as synthetic glycerol.

Fermentation and Harvesting The cultures of the working cell bank were used to inoculate seed bottles containing a soy-based medium. The bottles were incubated at 36 ° C ± 2 ° C without agitation until the growth requirements were met. A seed bottle was used to inoculate a seed fermentor containing soy-based medium. A pH of about 7.0 was maintained with sterile sodium carbonate solution. After the target optical density was reached, the seed fermenter was used to inoculate the production fermenter containing soy-based medium. The pH was maintained with sterile sodium carbonate solution. The fermentation was finished after growth stops or when the work volume of the fermenter was reached. An appropriate amount of sterile 12% sodium deoxycholate was added to the culture to lyse the bacterial cells and release of polysaccharide associated with the cell. After lysing, the contents of the fermenter were cooled. The pH of the lysate culture broth was adjusted to approximately pH 6.6 with acetic acid. The lysate was clarified by continuous flow centrifugation followed by deep filtration and 0.45 μm microfiltration.

In an alternate process, the pH fermentation of about 7.0 was maintained with 3N NaOH. After the objective optical density was reached, the sowing fermenter was used to inoculate the production fermenter containing soy-based medium. The pH was maintained with 3N NaOH. The fermentation was finished after growth stops or when the work volume of the fermenter was reached. An appropriate amount of sterile 12% deoxygenated sodium was added to the culture to obtain a 0.12% concentration in the broth, to lyse the bacterial cells and release polysaccharides associated with the cell. After lysing, the contents of the fermentor were maintained, with agitation, for a time interval between 8 and 24 hours at a temperature between 7 ° C and 13 ° C, to ensure that the complete cell lysis and the release of the polysaccharide were carried out. presented. Agitation during this waiting period prevents the sediment of the lysate from settling on the walls of the fermenter and pH probe, thereby allowing the integrity of the pH probe to be maintained. Then, the pH of the lysate broth was adjusted to about pH 5.0 with 50% acetic acid. After a waiting period without agitation, for a time interval between 12 and 24 hours at a temperature between 15 ° C and 25 ° C, a significant portion of the previously soluble proteins are removed from the solution as a solid precipitate with little loss or degradation of polysaccharide, which remains in the solution. The solution with the precipitate was then clarified by continuous flow centrifugation followed by deep filtration and 0.45 μm microfiltration.

Purification The purification of the pneumococcal polysaccharide consists of various concentration / diafiltration, precipitation / elution, column chromatography, and deep filtration steps. All procedures were performed at room temperature unless otherwise specified. The clarified broth from the cultures of the serotype 1 fermentor of S. pneumoniae were concentrated and diafiltered using a 100 kDa MWCO filter (kilodalton molecular weight limit). The diafiltration was carried out using a sodium phosphate buffer at a neutral pH. Diafiltration removes the low molecular weight components of high molecular weight biopolymers such as nucleic acid, protein and polysaccharide. The polysaccharide was precipitated from the concentrated and diafiltered solution by adding hexadecyltrimethyl ammonium bromide (HB) from a stock solution to give a final concentration of HB 1% (w / v). The polysaccharide / HB precipitate was captured in a deep filter and the filtered was discarded. The polysaccharide precipitate was resolubilized and eluted by recirculating a sodium chloride solution through the deep filter containing the precipitate. The filters were then rinsed with additional sodium chloride solution. Sodium iodide (Nal) was added to the polysaccharide solution of a reserve Nal solution to reach a final concentration of 0.5% to precipitate HB. The precipitate was removed by deep filtration. The filtrate contains the target polysaccharide. The precipitation vessel and the filter were rinsed with a NaCl / Nal solution and the rinse was combined with the partially purified polysaccharide solution. The filter was discarded. The polysaccharide was then filtered through a 0.2 μm filter. The polysaccharide solution was concentrated in a 30 kDa ultrafilter MWCO and diafiltered with a sodium chloride solution. The partially purified polysaccharide solution was further purified by filtration through a deep filter impregnated with activated carbon. After filtration, the carbon filter was rinsed with a sodium chloride solution. The rinse is combined with the polysaccharide solution, which is then filtered through a 0.2 μm filter. The polysaccharide solution was concentrated in a ultrafilter 30 kDa MWCO and adjusted with a 1 M sodium phosphate buffer solution to reach a final concentration of 0.025 M sodium phosphate. The pH was revised and adjusted to 7.0 ± 0.2. The ceramic hydroxyapatite (HA) column was equilibrated with sodium phosphate buffer solution containing sodium chloride until the appropriate conductivity was obtained (<15 μS). The polysaccharide solution was then loaded onto the column. Under these conditions, the impurities bound to the resin and the polysaccharide were recovered through the flow of the column. The polysaccharide solution was filtered through 0.2μm in-line filters located before and after the column. The polysaccharide solution was concentrated using a 30 kDa MWCO filter. The concentrate was then diafiltered with water for injection (WFI). The diafiltered polysaccharide solution was filtered through a 0.2 μm membrane filter in polypropylene bottles. The samples were removed by release tests and the purified polysaccharide was stored by freezing at -25 ° ± 5 ° C.

Characterization The 1H-NMR data were consistent with the chemical structure by the assignment of signals assigned to the protons of the polysaccharide molecule. The 1H-NMR spectrum shows a series of well resolved signals (protons of the methyl group) for the quantification of the functional group O-acetyl group in the polysaccharide. The identity of the monovalent polysaccharide was confirmed by countercurrent immunoelectrophoresis using specific antisera. High resolution gel filtration chromatography coupled with a refractive index and multiple angle laser light scattering detectors (MALLS) was used in conjunction with the sample concentration to calculate the molecular weight. The size exclusion chromatography medium (CL-4B) was used to profile the relative molecular size distribution of the polysaccharide.

Example 2 Preparation of Saccharide Conjugate of Serotype 1-Conjugate Pneumococcus Conjugate Activation and Conjugation Containers of the purified polysaccharide were thawed and combined in a reaction vessel. To the vessel, 0.2 M sodium carbonate, pH 9.0 was added for partial deacetylation (hydrolysis) for 3 hours at 50 ° C. The reaction was cooled to 20 ° C and the neutralization was performed by 0.2 M acetic acid. Oxidation in the presence of sodium periodate was performed by incubation at 2-8 ° C, and the mixture was stirred for 15-21 hours. The activation reaction mixture was concentrated and diafiltered by lOx with 0.9% NaCl using a 30K MWCO membrane. The retention product was 0.2 μm filtered. The activated saccharide was filled into glass lyophilization bottles of 100 mL and frozen in the envelope at -75 ° C and lyophilized. "Freezing in the envelope" is a method to prepare samples for lyophilization (freeze drying). The flasks are automatically rotated by motor driven rollers in a refrigerated bath containing alcohol and other appropriate fluid. A thin coating of the product is eventually frozen around the inner "shell" of a flask, which allows a larger volume of material to be processed safely during each freeze drying run. These refrigerated, automatic units provide a simple and efficient means of pre-freezing many flasks at one time, producing the desired interior coatings, and providing a sufficient surface area for efficient freeze drying. The bottles of lyophilized material are brought to room temperature and resuspended in CRM197 solution at a saccharide / protein ratio of 2: 1. To the mixture of saccharide / protein, a sodium phosphate buffer solution ΔM was added to a final 0.2M ion resistance and a pH of 7.5, then sodium cyanoborohydride was added. The reaction was incubated at 23 ° C for 18 hours, followed by a second incubation at 37 ° C for 72 hours. After incubations of cyanoborohydride, the reaction mixture was diluted with saline followed by the addition of sodium carbonate, IM to adjust the reaction mixture to pH 9.0. The unreacted aldehydes were quenched by the addition of sodium borohydride by incubation at 23 ° C for 3-6 hours. The reaction mixture was diluted 2 times with saline and transferred through a pre-filter of 0.45-5 μm in a container of retention product. The reaction mixture is diafiltered 30 × with 0.15 M phosphate buffer, pH 6, and 20 × with saline. The retention product was filtered through a 0.2 μm filter. The conjugate solution was diluted to a target of 0.5 mg / mL in 0.9% saline, and then sterile filtered in final volume concentrate (BCF) containers in a Class 100 hood. The conjugate was stored at 2-8 °. C.

Characterization Size exclusion chromatography medium (CL-4B) was used to profile the molecular size distribution relative of the conjugate. The identity of the conjugate was confirmed by the slot-blot assay using specific antiserum. Saccharide and protein concentrations were determined by the uric acid and Lowry assays, respectively. The ratio of saccharide to protein in the conjugate complex covalently linked was obtained by the calculation: μg / mL saccharide Ratio = μg / mL protein The O-acetyl Content was measured by the Hestrin method (Hestrin et al., J. Biol. Chem. 1949, 180, p.249). The concentration ratio of O-acetyl to total saccharide concentration gives μmoles of O-acetyl per mg of saccharide.

Example 3 Preparation of Serum 3 Capsular Polysaccharide of S. Pneumoniae Preparation of Master and Working Cell Banks Serotype 3 of S. pneumoniae was obtained from Dr. Robert Austrian, University of Pensilvana, Philadelphia, Pensilvana. For the preparation of the cell bank system, see Example 1.

Fermentation and Harvesting Cell work cell cultures were used for inoculated seed bottles containing soy-based medium. The bottles were incubated at 36 ° C ± 2 ° C without agitation until the requirements are met. A seed bottle was used to inoculate a seed fermenter containing soy-based medium. A pH of about 7.0 was maintained with sterile sodium carbonate solution. After the target optical density was reached, the seed fermentor was used to inoculate an intermediate seed fermenter. After the target optical density was reached, the intermediate seed fermenter was used to inoculate the production fermenter. The pH was maintained with sterile sodium carbonate solution. The fermentation was finished after the work volume of the fermentor was reached. An appropriate amount of sterile 12% sodium deoxycholate was added to the culture to lyse the bacterial cells and release the polysaccharide associated with the cells. After lysing, the contents of the fermenter were cooled. The pH of the lysate broth was adjusted to approximately pH 6.6 with acetic acid. The lysate was clarified by continuous flow centrifugation followed by deep filtration and 0.45 μm microfiltration.

Purification The purification of the pneumococcal polysaccharide consists of several operations of concentration / diafiltration, precipitation / elution, column chromatography, and deep filtration steps. All procedures were performed at room temperature unless otherwise specified. The clarified broth from the S. pneumoniae serotype 3 fermentor cultures was concentrated and diafiltered using a 100 kDa MWCO filter. The diafiltration was carried out using sodium phosphate buffered at neutral pH. Diafiltration removes the low molecular weight media components of the higher molecular weight biopolymers such as nucleic acid, protein and polysaccharide. Prior to the addition of hexadecyltrimethyl ammonium bromide (HB), a calculated volume of a NaCl stock solution was added to the concentrated and diafiltered polysaccharide solution to give a final concentration of 0.25 M NaCl. The polysaccharide was then precipitated by adding HB from a stock solution to give a final concentration of 1% HB (w / v). The polysaccharide / HB precipitate was captured in a deep filter and the filtrate was discarded. The polysaccharide precipitate was re-solubilized and eluted by recirculating a sodium chloride solution through the deep filter containing the precipitate. The filters were then rinsed with additional sodium chloride solution.

Sodium iodide (Nal) was added to the polysaccharide solution of a Nal stock solution to reach a final concentration of 0.5% to precipitate HB. The precipitate was removed by deep filtration. The filtrate contains the target polysaccharide. The precipitation vessel and the filter were rinsed with a NaCl / Nal solution and the rinse was combined with the partially purified polysaccharide solution. The filter was discarded. The polysaccharide was then filtered through a 0.2 μm filter. The polysaccharide solution was concentrated in a 30 kDa MWCO ultrafilter and diafiltered with a sodium chloride solution. The partially purified polysaccharide solution was further purified by filtration through a deep filter impregnated with activated carbon. After filtration, the carbon filter was rinsed with a sodium chloride solution. The rinsing was combined with the polysaccharide solution, which was then filtered through a 0.2 μm filter. The polysaccharide solution was concentrated in a 30 kDa MWCO ultrafilter and adjusted with a 1M sodium phosphate buffer to reach a final concentration of 0.025M sodium phosphate. The pH was revised and adjusted to 7.0 ± 0.2.

The ceramic hydroxyapatite (HA) column was equilibrated with sodium phosphate buffer solution containing sodium chloride to obtain the appropriate conductivity (15 μS). The polysaccharide solution was then loaded onto the column. Under these conditions, the impurities bound to the resin and the polysaccharide was recovered through the flow of the column. The polysaccharide was wetted through the column with buffer and filtered through a 0.2μm filter. The polysaccharide solution was concentrated using a 30 kDa MWCO filter. The concentrate was then diafiltered with WFI. The diafiltered polysaccharide solution was filtered through a 0.2 μm membrane filter in stainless steel containers. Samples were removed to test the release and the purified polysaccharide was stored frozen at -25 ° ± 5 ° C.

Characterization The 1H-NMR data were consistent with the chemical structure by the assignment of signals assigned to the protons of the polysaccharide molecule. The identity of the monovalent polysaccharide was confirmed by countercurrent immunoelectrophoresis using specific antiserum.

High performance gel filtration chromatography, coupled with refractive index and multiple angle laser light scattering detectors (MALLS), was used in conjunction with the sample concentration to calculate molecular weight. The size exclusion chromatography medium (CL-4B) was used to profile the relative molecular size distribution of the polysaccharide.

Example 4 Preparation of Serotype 3 Conjugate of Pneumococcal Saccharide -CRM197 Activation and Conjugation Serotype 3 purified saccharide containers were thawed and combined in a reaction vessel. To the vessel, WFI and 2M acetic acid were added to a final concentration of 0.2M and 2mg / mL saccharide. The temperature of the solution was raised to 85 ° C for one hour to hydrolyze the polysaccharide. The reaction was cooled to < 25 ° C and 1 M magnesium chloride was added to a final concentration of 0.1 M. Oxidation in the presence of sodium periodate was performed by incubation for 16-24 hours at 23 ° C. The activation reaction mixture was concentrated and diafiltered lOx with WFI using a 100K MWCO membrane. The retention product was filtered through a 0.2- filter. μm. For compound formation, 0.2M sodium phosphate, pH 7.0, was added to the activated saccharide at a final concentration of 10mM and a pH of 6.0-6.5. the protam protein CRM? 97 was mixed with the saccharide solution at a ratio of 2 g of saccharide per l of CRM? 97. The combined saccharide / protein solution was filled into 100 mL glass lyophilization bottles with a 50 mL objective fill, frozen in the envelope at -75 ° C, and lyophilized. The bottles of saccharide material / co-lyophilized protein are brought to room temperature and resuspended in 0.1 M sodium phosphate buffer, pH 7.0, to a final saccharide concentration of 20 mg / mL. The pH was adjusted to 6.5 and then a 0.5 molar equivalent of sodium cyanoborohydride was added. The reaction was incubated at 37 ° C for 48 hours. After incubation of cyanoborohydride, the reaction mixture was diluted with cold 5mM succinate / 0.9% buffered saline. The unreacted aldehydes were quenched by the addition of sodium borohydride and incubation at 23 ° C for 3-6 hours. The reaction mixture was transferred through a prefilter of 0.45-5 μm in a container of retention product. The reaction mixture was diafiltered 30x with 0.1 M phosphate buffer solution (pH 9), 20x with 0.15M solution phosphate buffer (pH 6), and 20x with 5mM succinate / O .9% saline. The retention product was filtered through a 0.2-μm filter. The conjugate solution was diluted to a saccharide target of 0.5 mg / mL, and then sterile filtered in FBC vessels in a Class 100 hood. The conjugate was stored at 2-8 ° C.

Characterization The size exclusion chromatography medium (CL-4B) was used to profile the relative molecular size distribution of the conjugate. The identity of the conjugate was confirmed by the slit-spot assay using specific antiserum. Saccharide and protein concentrations were determined by the Anthrone 10 and Lowry assays, respectively. The ratio of saccharide to protein in the conjugated complex covalently linked was obtained by calculation: μg / mL saccharide Ratio = μg / mL protein Example 5 Preparation of Serotype 5 Capsular Polysaccharide of S. Pneumoniae Serotype 5 of S. Pneumoniae was obtained from Dr. Gerald Schiffman of the State University of New York, Brooklyn, NY. For the preparation of the cell bank system, see Example 1. For fermentation, harvesting, purification and characterization of the polysaccharide, see Example 1.

Alternative Fermentation Processes The cell work cell cultures were used for inoculated seed bottles containing a soy-based medium and a sterile NaHCO 3 solution. The bottles were incubated at 36 ° C ± 2 ° C without agitation until the requirements are met. A seed bottle was used to inoculate a seed fermenter containing soy-based medium and a sterile NaHC03 lOmM solution. A pH of about 7.0 was maintained with 3N NaOH. After the target optical density was reached, the seed fermenter was used to inoculate the production fermentor containing soy-based medium with an lOmM concentration of NaHCO 3. The pH was maintained with 3N NaOH. The fermentation was finished after growth stops or when the work volume of the fermenter was reached. An appropriate amount of sterile 12% sodium deoxycholate was added to the culture to obtain a 0.12% concentration in the broth, to lyse the bacterial cells and release the polysaccharide associated with the cells. After lysing, the contents of the fermentor are maintained, with agitation, for a time interval between 8 and 24 hours at a temperature between 7 ° C and 13 ° C to ensure that the complete cell lysis and release of the polysaccharide have occurred. . Agitation during this waiting period prevents a sediment of lysis from settling on the walls of the fermenter and pH probe, thereby allowing the integrity of the pH probe to be maintained. Then, the pH of the lysate broth was adjusted to about pH 4.5 with 50% acetic acid. After a waiting period without agitation, for a time interval between 12 and 24 hours at a temperature between 15 ° C and 25 ° C, a significant portion of the previously soluble proteins are removed from the solution as a solid precipitate with little loss or degradation of the polysaccharide, which remains in the solution. The solution with the precipitate was then clarified by continuous flow centrifugation followed by deep filtration and 0.45 μm microfiltration.

Example 6 Preparation of Saccharide Pneumococcal Conjugate of Serotype 5 - CRM197 Activation and Conjugation Serotype 5 saccharide recipients were thawed and combined in a reaction vessel. To the vessel, 0.1 M sodium acetate, pH 4.7, was added followed by oxidation in the presence of sodium periodate by incubation for 16-22 hours at 23 ° C. The activation reaction mixture was concentrated and diafiltered lOx with WFI using a 100K MWCO membrane. The retention product was filtered through a 0.2 μm filter. The activated serotype 5 saccharide was combined with CRM197 at a ratio of 0.8: 1. The combined saccharide / protein solution was filled into 100 mL glass lyophilization bottles (50 mL target filling), frozen in the shell at 75 ° C, and mixed. The bottles of co-ground material are brought to room temperature and resuspended in 0.1 M sodium phosphate, pH 7.5, and sodium cyanoborohydride is added. The reaction was incubated at 30 ° C for 72 hours, followed by a second addition of cyanoborohydride and incubated at 30 ° C for 20-28 hours. After the cyanoborohydride incubations, the reaction mixture was diluted 2 times with saline and transferred through a 0.45-5 μm prefilter into a retention product container. The reaction mixture was diafiltered 30x with 0.01 M phosphate buffer, pH 8, 20x with 0.15M phosphate buffer, pH 6, and 20x with saline. The retention product was filtered through a 0.2 μm filter. The conjugate solution was diluted to a saccharide target of 0.5 mg / mL, and then sterile filtered in FBC vessels in a Class 100 hood. The conjugate was stored at 2-8 ° C. For the characterization of the conjugate, see Example 2.

Example 7 Preparation of Serotype 6A of Capsular Polysaccharide of S. Pneumoniae Serotype 6A S. pneumoniae was obtained from Dr. Gerald Schiffman of the State University of New York, Brooklyn, New York. For the preparation of the cell banking system, see Example 1. For fermentation, harvesting and purification of the polysaccharide, see Example 1, except that during purification, the 30 kDa MWCO concentration step, before the chromatographic step, is omitted.

Example 8 Preparation of Penumococcal Sacumoido Serotype 6A-CRM Conjugate 97 Activation and Conjugation The polysaccharide serotype 6A is a high weight polymer molecular that reduces in size before oxidation. Serotype 6A saccharide containers were thawed and combined in a reaction vessel. To the vessel, 2 M acetic acid was added to a final concentration of 0.1 M for hydrolysis for 1.5 hour at 60 ° C. The reaction was cooled to 23 ° C and neutralization was performed by adjusting the reaction mixture with 1 M NaOH to pH 6. Oxidation in the presence of sodium periodate was performed by incubation at 23 ° C for 14-22 hours. The activation reaction mixture was concentrated and diafiltered lOx with WFI using a 100K MWCO membrane. The retention product was filtered through a 0.2 μm filter. Serotype 6A was made in compound with sucrose and filled into 100 mL glass lyophilization bottles (50mL target filling) and frozen in the envelope at -75 ° C and lyophilized. The bottles of lyophilized material are brought to room temperature and resuspended in dimethylsulfoxide (DMSO) at a saccharide / protein ratio of 1: 1. After the addition of sodium cyanoborohydride, the reaction mixture was incubated at 23 ° C for 18 hours. After incubation of cyanoborohydride, the reaction mixture was diluted with cold saline. The unreacted aldehydes were quenched by the addition of sodium borohydride by incubation at 23 ° C for 3-20 hours.

The diluted reaction mixture was transferred through a 5 μm prefilter into a retention product container. The reaction mixture was diafiltered lOx with 0.9% NaCl and 30x with NaCl buffered in succinate. The retention product was filtered through a 0.2 μm filter. The conjugate solution was diluted to a saccharide target of 0.5 mg / mL, and then sterile filtered in FBC vessels in a Class 100 hood. The conjugate was stored at 2-8 ° C. For the characterization of the conjugate, see Example 2.

Example 9 Preparation of Serotype 7F of Capsular Polysaccharide of S. Pneumoniae Serotype 7F of S. pneumoniae was obtained from Dr. Gerald Schiffman of the State University of New York, Brooklyn, New York. For the preparation of the cell bank system, and for fermentation and harvesting of the polysaccharide, see Example 3. For an alternative fermentation and harvesting process, see the alternative process described in Example 1.

Purification The purification of the pneumococcal polysaccharide consists of several concentration / diafiltration, precipitation / elution, column chromatography, and steps of deep filtration. All procedures were performed at room temperature unless otherwise specified. The clarified broth of the fermenter cultures of serotype 7F of S. pneumoniae were concentrated and diafiltered using a 100 kDa MWCO filter. The diafiltration was carried out using sodium phosphate buffered at neutral pH. Diafiltration removes the low molecular weight media components of the higher molecular weight biopolymers such as nucleic acid, protein and polysaccharide. Serotype 7F does not form a precipitate with HB. On the contrary, the impurities were precipitated from the concentrated and diafiltered solution by adding the HB of a stock solution to a final concentration of 1% HB. The precipitate was captured in a deep filter and the filter was discarded. The polysaccharide was contained in the filtrate. Sodium iodide (Nal) was added to the polysaccharide solution of a Nal stock solution to reach a final concentration of 0.5% to precipitate HB. The precipitate was removed by deep filtration. The filtrate contains the target polysaccharide. The precipitation vessel and the filter were rinsed with a NaCl / Nal solution and the rinses were combined with the partially purified polysaccharide solution. The filter was discarded. The polysaccharide was then filtered through a 0.2μm filter.

The polysaccharide solution was concentrated in a 30 kDa MWCO ultrafilter and diafiltered with a sodium chloride solution. The partially purified polysaccharide solution was further purified by filtration through a deep filter impregnated with activated carbon. After filtration, the carbon filter was rinsed with a sodium chloride solution. The rinsing was combined with the polysaccharide solution, which was then filtered through a 0.2 μm filter. The polysaccharide solution was concentrated in a 30 kDa MWCO ultrafilter and adjusted with a 1M sodium phosphate buffer to reach a final concentration of 0.025M sodium phosphate. The pH was checked and adjusted to 7.0 ± 0.2. The ceramic hydroxyapatite (HA) column was equilibrated with sodium phosphate buffer containing sodium chloride to obtain the appropriate conductivity (15 μS). The polysaccharide solution was then loaded onto the column. Under these conditions, the impurities bound to the resin and the polysaccharide were recovered through the flow of the column. The polysaccharide was wetted through the column with buffer and filtered through a 0.2 μm filter. The polysaccharide solution was concentrated using a 30 kDa MWCO filter. The concentrate was then diafiltered with WFI The diafiltered polysaccharide solution was filtered through a 0.2 μm membrane filter in stainless steel containers. Samples were removed to test the release and the purified polysaccharide was stored at 2 ° -8 ° C. For the characterization of the polysaccharide, see Example 3.

Example 10 Preparation of Serotype 7F-CRM Saccharide Saccharide Conjugate 97 Activation and Conjugation Oxidation in the presence of sodium periodate was performed by incubation for 16-24 hrs at 23 ° C. The activation reaction mixture was concentrated and diafiltered by lOx with lOmM NaOAc, pH 4.5, using a 100K MWCO membrane. The retention product was filtered through a 0.2 μm filter. Serotype 7F was filled into 100 mL glass lyophilization bottles (50 ml, objective filler) and frozen in the envelope at -75 ° C and lyophilized. The lyophilized serotype 7F and CRM? 9 bottles are brought to room temperature and resuspended in DMSO at a saccharide / protein ratio of 1.5: 1. After the addition of sodium cyanoborohydride, the reaction was incubated at 23 ° C for 8-10 hours. The unreacted aldehydes are were quenched by the addition of sodium borohydride by incubation at 23 ° C for 16 hours. The reaction mixture was diluted 10 times with saline and transferred through a 5 μm prefilter into a container of retention product. The reaction mixture was diafiltered lOx with 0.9% saline and 30x with saline buffered in succinate. The retention product was filtered through a 0.2 μm filter. The conjugate solution was diluted to a saccharide target of 0.5 mg / mL 0.9% saline, and then sterile filtered in FBC vessels in a Class 100 hood. The conjugate was stored at 2-8 ° C. For the characterization of the conjugate, see Example 4.

Example 11 Preparation of serotype 19A of the capsular polysaccharide of S. pneumoniae Serotype 19A of S. pneumoniae was obtained from Dr. Gerald Schiffman of the State University of New York, Brooklyn, New York. For the preparation of the cell bank system, see Example 1. For fermentation, harvesting and purification of the polysaccharide, see Example 7. For characterization, see Example 3.

Example 12 Preparation of Pneumococcal Saccharide Conjugate of Serotype 19A-CRM 97 Activation and Conjugation The serotype 19A saccharide containers were thawed and combined in a reaction vessel. Sodium acetate was added at 10 mM (pH 5.0) and oxidation was carried out in the presence of sodium periodate by incubation for 16-24 hrs at 23 ° C. The activation reaction mixture was concentrated and dialyzed lOx with lOmM acetate, pH 5.0, using a 100K MWCO membrane. The retention product was filtered through a 0.2 μm filter. The activated saccharide was made in compound with sucrose followed by the addition of CRM197. The activated saccharide mixture of serotype 19A and CR 197 (ratio 0.8: 1) was filled into 100 mL glass lyophilization bottles (50 mL target filling) and frozen in the envelope at -75 ° C and lyophilized. The bottles of lyophilized material are brought to room temperature and resuspended in DMSO. To the saccharide / protein mixture, sodium cyanoborohydride (100 mg / ml) was added. The reaction was incubated at 23 ° C for 15 hours. After incubation of cyanoborohydride, the unreacted aldehydes were quenched by the addition of sodium borohydride by incubation at 23 ° C for 3-20 hours. The reaction mixture was diluted 10 times with saline and transferred through a 5 μm prefilter into a container of retention product. The reaction mixture was diafiltered by lOx with 0.9% NaCl, filtered 0.45-μm, and 30x with diafiltration using 5 mM succinate buffer solution / 0.9% NaCl, pH 6. The retention product was filtered through a 0.2 μm filter. The conjugate solution was diluted to a target of 0.5 mg / mL using 5mM succinate / 0.9% saline, and then sterile filtered in FBC vessels in a Class 100 hood. The conjugate was stored at 2-8 ° C. For the characterization of the conjugate, see Example 4.

Example 13 Preparation of Serotypes 4, 6B, 9V, 14, 18C, 19F and 23F Capsular Polysaccharide of S. Pneumoniae Preparation of S-Seed Culture. pneumoniae Serotypes 4, 6B, 9V, 18CX 19F and 23F of S. pneumoniae were obtained from Dr. Gerald Schiffman, State University of New York, Brooklyn, New York. Serotype 14 of S. pneumoniae was obtained from the ATCC, strain 6314. Separately, one vial of each of the desired serotypes of Streptococcus pneumoniae was used to initiate a fermentation batch. Two bottles containing a medium based on soybean and red phenol were adjusted to a pH range of 7.4 ± 0.2 using sodium carbonate, and the required volume of 50% dextrose solution / 1% magnesium sulfate was then added to the bottles The two bottles were inoculated with different amounts of seed. The bottles were incubated at 36 ° ± 2 ° C until the medium turns yellow. After incubation, the samples were removed from each bottle and tested for optical density (OD) (0.3 to 0.9) and pH (4.6 to 5.5). One of the two bottles was selected for inoculation of the sowing fermenter. The soy-based medium was transferred to the sowing fermenter and sterilized. Then, a volume of 50% dextrose solution / 1% magnesium sulfate was added to the fermenter. The pH and agitation of the seed fermenter were monitored and controlled (pH 6.7 to 7.4). The temperature was maintained at 36 ° ± 2 ° C. The seed inoculate (bottle) was aseptically connected to the seed fermenter and the inoculum was transferred. The fermentor was maintained in pH control and the samples were periodically removed and tested for OD and pH. When the desired OD of 0.5 to 600 nm was reached, the intermediate fermentor was inoculated with the fermentation broth from the sowing fermentor. The soy-based medium was transferred to the intermediate fermentor and sterilized. Then a volume of 50% dextrose solution / 1% magnesium sulfate was added to the fermenter. The pH and agitation of the intermediate fermentor were monitored and controlled (pH 6.7 to 7.4). The temperature was maintained at 36 ° ± 2 ° C. The contents of the sowing fermenter were transferred to the intermediate fermentor. The fermentor was kept in control of the pH and the samples were removed and tested periodically for OD and pH. When the desired OD of 0.5 to 600 nm was reached, the production fermenter was inoculated with the fermentation broth from the intermediate fermentor. The soy-based medium was transferred to the production fermenter and sterilized. Then a volume of 50% dextrose solution / 1% magnesium sulfate was added to the fermenter. The pH and agitation of the production fermenter were monitored and controlled (pH 6.7 to 7.4). The temperature was maintained at 36 ° ± 2 ° C. The fermentor was kept in control of the pH and the samples were removed and tested periodically for OD and pH, until the fermentation was complete. Sodium deoxycholate was added to the fermentor at a final concentration of approximately 0.12% w / v. The culture was mixed for a minimum of thirty minutes and the temperature set point was reduced to 10 ° C. The culture was incubated overnight and confirmation of inactivation followed, the pH of the culture was adjusted to between 6.4 and 6.8, as necessary, with 50% acetic acid. Temperature The fermenter was increased to 20 ° ± 5 ° C and the contents were transferred to the clarification tank. The contents of the clarification tank (including cellular strands) were processed through a centrifuge at a flow rate between 25 and 600 liters per hour (except Serotype 4, where the cell strands were discarded and the ratio of flow narrowed to between 25 and 250 liters per hour). Samples of the supernatant were removed and tested for OD. The desired OD during the centrifugation was < 0.15. Initially, the supernatant was recirculated through a deep-seated filter until an OD of 0. 05 ± 0.03 was reached. The supernatant was then passed through the assembled deep filter and through a 0.45 μm membrane filter to the filter stand-by tank. Subsequently, the product was transferred through closed tubes to the purification area for processing. All the previous operations (centrifugation, filtration and transfer) were carried out between 10 ° C to 30 ° C. For an alternative fermentation and harvesting process for serotypes 4 and 6B, see the alternative process described in Example 1.

Purification The purification of each pneumococcal polysaccharide consists of several operations of concentration / diafiltration, precipitation / elution, column chromatography, and deep filtration steps. All procedures were performed at room temperature unless otherwise specified. The clarified broth from the cultures of the fermenter of the desired S. pneumoniae serotype was concentrated and diafiltered using a 100 kDa MWCO filter. The diafiltration was performed using sodium phosphate buffer solution at pH < 9.0. Diafiltration removes the low molecular weight medium components of the higher molecular weight biopolymers such as nucleic acid, protein and polysaccharide. The polysaccharide was precipitated from the concentrated and diafiltered solution by adding HB from a stock solution to give a final concentration of 1% HB (w / v) (except Serotype 23F, which has a final concentration of 2.5%). The polysaccharide / HB precipitate was captured in a deep filter and the filtrate was discarded. (Note: serotype 14 does not precipitate, therefore the filtrate was maintained.) The polysaccharide precipitate was re-solubilized and eluted by recirculating a sodium chloride solution through the deep filter containing the precipitate. The filters were then rinsed with sodium chloride solution additional . Sodium iodide (Nal) was added to the polysaccharide solution of a reserve Nal solution to reach a final concentration of 0.5% to precipitate HB (except for Serotype 6B, which has a final concentration of 0.25%). The precipitate was removed by deep filtration. The filtrate contains the target polysaccharide. The filter was discarded. The polysaccharide was then filtered through a 0.2μm filter. The polysaccharide solution was concentrated in a 30 kDa MWCO ultrafilter and diafiltered with a sodium chloride solution. The partially purified polysaccharide solution was further purified by filtration through a deep filter impregnated with activated carbon. After filtration, the carbon filter was rinsed with a sodium chloride solution. The rinsing was combined with the polysaccharide solution, which was then filtered through a 0.2μm filter. The polysaccharide solution was concentrated in a 30 kDa ultrafilter MWCO and the filter was rinsed with a sodium chloride solution. The pH was checked and adjusted to 7.0 ± 0.3. The ceramic hydroxyapatite (HA) column was equilibrated with sodium phosphate buffer containing Sodium chloride until the pH is 7.0 ± 0.3 and the conductivity was 26 + 4μS. The polysaccharide solution was then loaded onto the column. Under these conditions, the impurities bound to the resin and the polysaccharide were recovered in the flow through the column. The polysaccharide solution was filtered through a 0.2μm filter. The polysaccharide solution was concentrated using a 30 kDa MWCO filter. The concentrate was then diafiltered with WFI until the conductivity was < 15μS. The diafiltered polysaccharide solution was filtered through a 0.2μm membrane filter in volume containers and stored at 2-8 ° C.

Example 14 Preparation of Saccharide Conjugates of Pneumococcus - CRM? 97 for Serotypes 4, 6B, 9V, 14, 18C, 19F and 23F Activation Process The different saccharide serotypes follow different trajectories for activation (hydrolysis or without hydrolysis before activation) and conjugate (water or DMSO reactions) as described in this example. The polysaccharide was transferred from the volume recipients to the reactor vessel. The polysaccharide was then diluted in WFI and sodium phosphate to a final concentration in the range of 1.6-2.4 mg / mL Stage 1 . For serotypes 6B, 9V, 14, 19F and 23F, the pH was adjusted to pH 6.0 ± 0.3. For serotype 4, hydrochloric acid (final acid concentration 0.01 M) was added and the solution was incubated for 25-35 minutes at 45 ° ± 2 ° C. The hydrolysis was stopped by cooling to 21-25 ° C and adding sodium phosphate ÍM to a target pH 6.7 ± 0.2. An ongoing test is given to confirm an appropriate level of de-waxing. For serotype 18C, glacial acetic acid (0.2 M final acid concentration) was added and the solution was incubated for 205-215 minutes at 94 ° ± 2 ° C. The temperature was then reduced to 21-25 ° C and 1-2 M sodium phosphate was added to a target pH 6.8 ± 0.2.

Step 2: Reaction of Periodate to The molar equivalents of sodium periodate required for the activation of pneumococcal saccharide was determined using total saccharide content (except for serotype 4). For serotype 4, a ratio of 0.8-1.2 moles of sodium periodate per mole of saccharide was used. With thorough mixing, the oxidation reaction was allowed to proceed between 16 to 20 hours at 21-25 ° C for all serotypes except 19F for which the temperature was = 15 ° C.

Stage 3: Ul traction The oxidized extraction was concentrated and diafiltered with WFI (0.01 M sodium phosphate buffer solution pH 6.0 for serotype 19F) in a 100 kDa ultrafilter MWCO (5 kDa ultrafilter for 18C). The permeate was discarded and the retention product was filtered through a 0.22 μm filter.

Stage 4: Lyophilization For serotypes 4, 9V, and 14, the concentrated saccharide was mixed with protein CRM? 97 protein, filled in glass bottles, frozen in the envelope and stored at < -65 ° C. The frozen concentrate-CRMi97 saccharose was lyophilized and then stored at -25 ° ± 5 ° C. For serotypes 6B, 19F, and 23F a specific amount of sucrose was added which was calculated to achieve a concentration of 5% ± 3% sucrose in the conjugation reaction mixture. Serotype 18C does not require the addition of sucrose. The concentrated saccharide was then filled into glass bottles, frozen in the shell and stored at < -65 ° C. The frozen concentrated saccharide was lyophilized and then stored at -25 ° ± 5 ° C.

Conjugation Process Two conjugation processes were used: aqueous conjugation for serotypes 4, 9V, 14 and 18C, and DMSO conjugation for serotypes 6B, 19F and 23F.

Aqueous Conjugation Stage 1: Di sol ution For serotypes 4, 9V and 14, the lyophilized activated CRM? 97-saccharide mixture was thawed and equilibrated at room temperature. The freeze-dried activated CRMi97 saccharide was then reconstituted in 0.1 M sodium phosphate buffer solution at a typical ratio of: 1 L of buffer solution for 16-24 g of saccharide for serotype 4 and 9V ° 1 L of buffer for 6 - 10 g of saccharide for serotype 14 The reaction mixture was incubated at 37 ° ± 2 ° C until the total solution for serotype 9V and at 23 ° ± 20C for serotypes 4 and 14. For serotype 18C, the lyophilized saccharide it was reconstituted in a solution of CRM197 in 1 M dibasic sodium phosphate at a typical ratio of 0.11 L sodium phosphate per 1 L of the CRM197 solution. The reaction mixture (8-12 g / L saccharide concentration) was incubated at 23 ° + 2 ° C until total dissolution. The pH was tested as in the control process at this stage.

Stage 2: Conjugal reaction For serotypes 4 and 9V, the conjugation reaction was initiated by adding the sodium cyanoborohydride solution (100 mg / mL) to reach 1.0-1.4 moles of sodium cyanoborohydride per mole of saccharide. The reaction mixture was incubated for 44-52 hours at 37 ° ± 2 ° C. The temperature was then reduced to 23 ° ± 2 ° C and 0.9% sodium chloride was added to the reactor. The sodium borohydride solution (100 mg / mL) was added to achieve 1.8 - 2.2 molar equivalents of sodium borohydride per mole of saccharide. The mixture was incubated for 3-6 hours at 23 ° ± 2 ° C. The mixture was diluted with 0.9% sodium chloride and the reactor was rinsed. The diluted conjugate mixture was filtered using a pre-filtered 1.2 μm in a maintenance vessel. For serotypes 14 and 18C, the conjugation reaction was initiated by adding the cyanoborohydride solution (100 mg / mL) to reach 1.0-1.4 moles of sodium cyanoborohydride per mole of saccharide. The reaction mixture was incubated for 12-24 hours at 23 ° ± 2 ° C. The temperature was increased to 37 ° ± 2 ° C and the reaction was incubated for 72-96 hours. The temperature was then reduced to 23 ° ± 2 ° C and 0.9% sodium chloride was added to the reactor. The sodium borohydride solution (100mg / mL) was added to achieve 1.8-2.2 molar equivalents of sodium borohydride per mole of saccharide. The mixture was incubated for 3-6 hours at 23 ° ± 2 ° C. The mixture was diluted with 0.9% Sodium chloride and the reactor was rinsed. The diluted conjugate mixture was then filtered using a 1.2 μm pre-filter in a maintenance vessel.

Stage 3: Ul trafil tra tion 100 kDa The diluted conjugate mixture was concentrated and diafiltered in a 100 kDa ultrafilter MWCO with either a minimum of 15 volumes (serotype 4) or 40 volumes (serotypes 9V, 14, and 18C) of 0.9 % sodium chloride. The permeate was discarded. For serotype 4, the retention product was filtered through a 0.45μm filter. A control in the process (saccharide content) was carried out in this stage.

Stage 4: Purification of the HA column This stage was only carried out by the serotype 4 conjugate. The HA column was first neutralized using 0.5M sodium phosphate buffer (pH 7.0 + 0.3) and then equilibrated with 0.9% sodium chloride. The filtered retention product (serotype 4) was loaded into the column at a flow rate of 1.0 L / min. The column was washed with 0.9% sodium chloride at a flow ratio of < 2.0 L / min. The product was then eluted with 0.5M sodium phosphate buffer at a flow ratio of < 2.0 L / min. The HA fraction was then concentrated and diafiltered in a 100 kDa MWCO membrane with a minimum of 20 volumes of 0.9% sodium chloride. The permeate was discarded.

Step 5: Sterile Filtration The retention product after 100 kDa MWCO of diafiltration was filtered through a 0.22 μm filter. In the process controls (saccharide content, free protein, free saccharide and cyanide) were carried out in the filtered product. In the process controls in the filtered retention product were carried out to determine if additional concentration, diafiltration, and / or dilution were necessary to meet the BCF objectives. These and additional tests were repeated in the FBC samples. As necessary, the filtered conjugate was diluted with 0.9% sodium chloride in order to reach a final concentration of less than 0.55 g / L. The release tests for the saccharide content, content of the ratio of the protein and saccharide: protein were carried out in this stage. Finally, the conjugate was filtered (0.22 μm) and filled in 10 L stainless steel canisters to a typical amount of 2.64 g / pot. In this stage, the production, saccharide content, protein content, pH, saccharide: protein ratio and lysine content was performed as in the process controls. The release test (appearance, free protein, free saccharide, endotoxin, determination of molecular size, residual cyanide, saccharide identity, CRM identity? 97) was performed in this stage.

DMSO Conjugation Stage I: Di sol ution The lyophilized activated saccharide serotypes 6B, 19F, 23F and lyophilized CR 197 carrier protein were equilibrated at room temperature and reconstituted in DMSO. The concentration of solution is typically in the range of 2-3 grams of saccharide (2-2.5 g protein) per liter of DMSO.

Stage II: Conjugation Reaction The activated saccharide and CRMi97 carrier protein were mixed for 60-75 minutes at 23 ° ± 2 ° C in a ratio range of 0.6 g - 1.0 g saccharide / g CRM197 for the serotypes 6B and 19F or 1.2 to 1.8 g saccharide / g CRM? 9 for serotype 23F. The conjugation reaction was initiated by adding the sodium cyanoborohydride solution (lOOmg / ml) at a ratio of 0.8-1.2 molar equivalents of sodium cyanoborohydride to one mole of activated saccharide. The WFI was added to the reaction mixture at a target of 1% (v / v) and the mixture was incubated for 40 hours at 23 ° ± 2 ° C. The sodium borohydride solution, 100 mg / mL (typical of 1.8-2.2 molar equivalents of sodium borohydride per mole of activated saccharide) and WFI (target 5% v / v) were added to the reaction and the mixture was incubated for 3 - 6 hours at 23 ° ± 2 ° C. This procedure reduces any unreacted aldehyde present in the saccharides. Then the reaction mixture was transferred to a dilution tank containing 0.9% sodium chloride at < 15 ° C.

Stage III: 100 kDa ultrafiltration The mixture of the diluted conjugate was filtered through a 1.2 μm filter and concentrated and diafiltered in a membrane. 100 kDa MWCO with a minimum of 15 volumes of 0.9% sodium chloride (0.01 M sodium phosphate / 0.05M buffer solution NaCl was used for serotype 23F). The permeate was discarded.

The retention product is filtered through a 0.45 μm filter. A sample of saccharide content in process was taken at this stage.

Stage IV: Purification of the DEAE column This stage was performed only for serotype 23F. The DEAE column was equilibrated with 0.01 M sodium phosphate / 0.05M sodium chloride buffer. The filtered retention product (serotype 23F) was loaded in column and was washed with 0.01 M sodium phosphate / 0.05M sodium chloride buffer solution. The column was then washed with 0.01 M sodium phosphate / 0.9% NaCl buffer. The product was then eluted with 0.01 M sodium phosphate / 0.5M sodium chloride buffer.

Step V: 100 kDa trafil trafil The retention product of 6B and 19F was concentrated and diafiltered with at least 30 volumes of 0.9% sodium chloride. The permeate was discarded. Eluate of serotype 23F was concentrated and diafiltered with a minimum of 20 volumes of 0.9% sodium chloride. The permeate was discarded.

Step VI: Sterile Filtration The retention product after the 100 kDa diafiltration MWCO was filtered through a 0.22 μm filter. In the process controls (saccharide content, free protein, free saccharide, residual DMSO and residual cyanide) were carried out in the filtrate. In the process controls in the filtered retention product were performed to determine if additional concentration, diafiltration, and / or dilution were necessary to meet the BCF objectives. These and additional tests were repeated in the samples FBC. As necessary, the filtered conjugate was diluted with 0. 9% sodium chloride to reach a final concentration of less than 0.55 g / L. Release tests for saccharide content, protein content and saccharide: protein ratio were performed in this stage. Finally, the conjugate was filtered (0.22 μm) and filled in 10 L stainless steel cans at an amount of 2.64 g / pot. In this stage, the production, saccharide content, protein content, pH, saccharide: protein ratio and lysine content was carried out as in the process controls. The release test (appearance, free protein, free saccharide, endotoxin, determination of molecular size, residual cyanide, residual DMSO, identity of saccharide and Identity CRM197) was carried out in this stage.

EXAMPLE 15 Formulation of a vaccine of the pneumococcal conjugate. The final volume concentrates of 13 conjugates containing 0.85% sodium chloride. Volume concentrates type 3, 6A, 7F and 19A also contain 5 mM sodium succinate buffer solution at pH 5.8. The required volumes of the volume concentrates were calculated based on the volume of the batch and the concentration volume of saccharides. After 80% of the 0.85% sodium chloride (physiological saline) and the required amount of succinate buffer were added to the pre-labeled formulation container, volume concentrates were added. The preparation was then sterile filtered through a 0.22 μm membrane in a second container by using a Millipore Durapore membrane filter unit. The first container was washed with the remaining 20% of 0.85% sodium chloride and the solution was passed through the same filter and collected in the second container. The volume of the formula was mixed gently during and following the addition of the volume of aluminum phosphate. The pH was checked and adjusted if necessary. The product of the formulated volume was stored at 2-8 ° C. The product of the formulated volume was filled into Type 1 borosilicate glass syringes obtained from Becton Dickinson. The vaccine was checked at regular intervals for turbidity to measure the uniformity of the filling operation. The full vaccine (final product) was stored at 2-8 ° C.

Example 16 Immunogenicity of the 1-valent conjugate vaccine To date, preclinical studies performed on the 13vPnC vaccine are done in rabbits. Studies # HT01-0021 and # HT01-0036 were designed to independently examine the chemical conjugation effect of capsular polysaccharides (PSs) from S. pneumoniae to CRM? 97 and the effect of aluminum phosphate adjuvant (AIP0) on the immune response to the 13vPnC vaccine in rabbits. These effects were characterized by the antigen-specific ELISA for the serum IgG concentrations and for the function of the antibody by the opsonophagocytic assay (OPA).

Study # HT01 -0021 Study # HT01-0021 examined the ability of the 13vPnC vaccine with the adjuvant AIP04 to produce the immune responses specific to the vaccine serotype. The pneumococcal serotypes represented in the 13vPnC vaccine include types 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C1 19A, 19F and 23F. Secondarily the objectives include an evaluation of the kinetics and duration of the response to the antibody. White New Zealand rabbits were immunized intramuscularly at week 0 and week 2 with the planned human clinical doses of each polysaccharide (2 μg of each PS, except 4 μg of 6B) were formulated with or without AIP04 (100 μg / dose) . The sera were collected at various time points. Serotype-specific IgG was measured by ELISA and functional activity was determined by OPA. Table 3 shows the geometric medium concentration (GMT) reached in the accumulated serum samples, following the two doses of the 13vPnC vaccine. A ratio of IgG GMTs it was used to compare the responses from week 4 to week 0. These data demonstrated that the inclusion of AIP04 in the 13vPnC formulation determines the highest levels of the IgG antibody compared to the same vaccine without adjuvant. Although the antibody responses are greater than AIP04 are included in the formulation, these increases are not statistically significant. Functional antibody responses are also determined in rabbits following immunization with the two 13vPnC formulations (Table 4). When comparing vaccine formulations with or without adjuvants, the OPA GMTs high was observed in the treatment group of the 13vPnC + AIP04 vaccine. OPA concentrations were detected in serum pools at week 4 for all serotypes of the vaccine in both groups. For most serotypes, the OPA concentrations measured at week 4 were at least 4-times higher than those at week 0 (baseline). The kinetic responses to each of the serotypes of the 13vPnC vaccine were evaluated from the serum groups of both treatment groups. The IgG concentrations at each serotype were measured from blood taken at week 0 and at weeks 1, 2, 3, 4, 8, 12, 26, and 39 and then compared. With the exception of serotype 1, the antibody responses in the animals receiving the adjuvant vaccine were superior to those receiving the vaccine without adjuvant and reached the peak in week 2 of the immunization program (data not shown). In general, the data indicate that the 13vPnC vaccine formulated with aluminum phosphate is immunogenic in rabbits, produces the response of the substantial antibody to the encapsulated pneumococcal polysaccharides containing the vaccine and these responses are associated with functional activity. The responses observed to the seven nuclei of serotypes following the immunization with 13vPnC + AIP04 consist of historical responses of rabbits to the heptavalent formulation.

Table 3. Rabbit IgG immune responses (GMTs) following immunization with two doses of the 13-valent pneumococcal glycoconjugate a: The GMTs of the pooled serum consist of equal volumes of serum from each individual rabbit within a group. b: Statistically different (p = 0.022) from the treatment group without ALP04.

Table 4. OPA GMT of S. pneumonias for NZ rabbit serum groups after immunization with two doses of 13-valent pneumococcal glycoconjugate A: Groups consist of equal volumes of serum from individual rabbits without a treatment group (n = 12) Study # HT01 -0036 Study # HT01-0036 compares the immune responses of rabbits to the polysaccharides (PSs) contained in the vaccine, after immunization with the 13vPnC vaccine with or without conjugation to the CRM protein? 97. The white rabbits of New Zeeland were immunized intramuscularly at week 0 and week 2 with a dose of 2.2 μg of each PS (except 4.4 μg of 6B). The animals received one of three vaccine preparations: (a) 13vPnC (PS directly conjugated to CRM? 97), (b) 13vPnPS, (free PS) or (c) 13vPnPS + CRM? 9 (free PS mixed with CRM 97). All preparations of the vaccine contain AIP04 as the adjuvant at 125 μg / dose. Serotype-specific immune responses for all vaccine preparations were evaluated in an IgG ELISA and functional antibody that measures complement-mediated OPA. The immune responses were compared between the treatment groups.

Table 5 presents the GMT data obtained from the bleeding at week 4 analyzed in the IgG ELISA specific antigen. Additional analyzes show a relationship of GMT values at week 4 through week 0. The data indicate that the preparation of the conjugate vaccine facilitates more serum IgG concentrations than free PS or free PS + CRM197 vaccine. With the exception of type 14 of S. pneumoniae, the 13vPnC vaccine was able to induce functional antibodies to the representative strains of S. pneumoniae in an OPA (Table 6). After the two immunizations with either the 13vPnPS or 13vPnPS + CRM197 vaccine, none could induce OPA concentrations>. 8 times in week 4 relative to week 0 for the 10 extractions of the 13 serotypes measured (Table 6) - In conclusion, these results indicate that the conjugation of the polysaccharides of the 13-valent pneumococcal vaccine produce higher serum IgG concentrations and in general greater activity of the functional antibody is observed with the free polysaccharide alone or mixed with the unconjugated CRM? 97.

Table 5. Rabbit IgG (GMT) responses for PnPS by ELISA After Immunization with Two Dose of 13-valent Pneumococcal Glycoconjugate Table 6. OPA concentrations of S. pneumoniae for the rabbit serum groups after immunization with two doses of 13-valent pneumococcal vaccine a: It is used as in the values in week 0 for all groups.

It should be understood that the above discussion and examples merely present a detailed description of certain modalities. This should also be apparent to those of ordinary skill in the art in various modifications and equivalents that can be made departing from the spirit and scope of the invention. All journal articles, other references, patents and patent applications identified in this patent application are incorporated for reference in their entirety.

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It is noted that in relation to this date, the best known method for carrying out the aforementioned invention is that which is clear from the present description of the invention.

Claims (26)

1. CLAIMS Having described the invention as above, the content of the following claims is claimed as property. A multivalent immunogenic composition, characterized in that it comprises: 13 different polysaccharide-protein conjugates, together with a physiologically acceptable carrier, wherein each of the conjugates comprises a capsular polysaccharide of a different serotype of Streptococcus pneumoniae conjugated to a carrier protein, and capsular polysaccharides are prepared from serotypes 1, 3, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F and 23F.
2. 2. The immunogenic composition according to claim 1, characterized in that the carrier protein is CRM197.
3. 3. The immunogenic composition according to claim 1, characterized in that it also comprises an adjuvant.
4. 4. The immunogenic composition according to claim 3, characterized in that the adjuvant is an aluminum-based adjuvant.
5. The immunogenic composition according to claim 4, characterized in that the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
6. 6. The immunogenic composition according to claim 5, characterized in that the adjuvant is aluminum phosphate.
7. 7. Use of an immunogenic composition according to claim 1 for the manufacture of a medicament for the induction of an immune response to a capsular polysaccharide conjugate of Streptococcus pneumoniae.
8. 8. Use according to claim 7, wherein the immunogenic composition administered is a single dose of 0.5 ml formulated to contain: 2 μg of each saccharide, except for 6B to 4 μg; approximately 29 μg of carrier protein CRN? 9; 0.125 mg of elemental aluminum adjuvant (0.5 mg of aluminum phosphate); and sodium chloride and sodium succinate buffer solution as excipients.
9. 9. A multivalent immunogenic composition, comprising polysaccharide-protein conjugates together with a physiologically acceptable carrier, characterized in that each of the conjugates comprises a capsular polysaccharide of a different serotype of Streptococcus pneumoniae conjugated to a carrier protein, and the capsular polysaccharides are prepare from serotype 3 and at least one additional serotype.
10. 10. The immunogenic composition in accordance with claim 9, characterized in that the additional serotype is selected from the group consisting of serotypes 1, 4, 5, 6A, 6B, 7F, 9V, 14, 18C, 19A, 19F, and 23F.
11. 11. The immunogenic composition according to claim 9, characterized in that the carrier protein is CRM? 97.
12. 12. The immunogenic composition according to claim 9, characterized in that it further comprises an adjuvant.
13. 13. The immunogenic composition according to claim 12, characterized in that the adjuvant is an aluminum-based adjuvant.
14. The immunogenic composition according to claim 13, characterized in that the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
15. 15. The immunogenic composition according to claim 14, characterized in that the adjuvant is aluminum phosphate.
16. 16. Use of an immunogenic composition according to claim 9, for the manufacture of a medicament for the induction of an immune response to a capsular polysaccharide conjugate of Streptococcus pneumoniae.
17. 17. Use according to claim 16, in wherein the immunogenic composition administered is a single dose of 0.5 ml. , formulated to contain: 2 μg of each saccharide, except for 6B to 4 μg; approximately 29 μg of carrier protein CRN? 97; 0.125 mg of elemental aluminum adjuvant (0.5 mg of aluminum phosphate); and sodium chloride and sodium succinate buffer solution as excipients.
18. 18. A multivalent immunogenic composition, comprising polysaccharide-protein conjugates together with a physiologically acceptable carrier, characterized by each conjugate comprising a capsular polysaccharide of a different serotype of Streptococcus pneumoniae conjugated to a carrier protein, and the capsular polysaccharides are prepared from serotypes 4, 6B, 9V , 14, 18C, 19F, 23F and at least one additional serotype.
19. 19. The immunogenic composition according to claim 18, characterized in that the additional serotype is selected from the group consisting of serotypes 1, 3, 5, 6A, 7F, and 19A.
20. The immunogenic composition according to claim 18, characterized in that the carrier protein is CRM197.
21. The immunogenic composition according to claim 18, characterized in that it also comprises an adjuvant.
22. 22. The immunogenic composition according to claim 21, characterized in that the adjuvant is an aluminum-based adjuvant.
23. 23. The immunogenic composition according to claim 22, characterized in that the adjuvant is selected from the group consisting of aluminum phosphate, aluminum sulfate and aluminum hydroxide.
24. 24. The immunogenic composition according to claim 23, characterized in that the adjuvant is aluminum phosphate.
25. 25. Use of an immunogenic composition according to claim 18, for the manufacture of a medicament for the induction of an immune response to a capsular polysaccharide conjugate of Streptococcus pneumoniae.
26. 26. Use according to claim 25, wherein the immunogenic composition administered is a single dose of 0.5 ml, formulated to contain: 2 μg of each saccharide, except for 6B to 4 μg; approximately 29 μg of carrier protein CRM? 97; 0.125 mg of elemental aluminum adjuvant (0.5 mg of aluminum phosphate); and sodium chloride and sodium succinate buffer solution as excipients.